We propose to study the replication of the resistance plasmid R100 in E. coli K12. The research is aimed at genetic and biochemical analysis of genes which are utilized in the replication of R1000. In the past year we have completed the nucleotide sequence of the region 2.5 kilobase pairs in length, which has been shown to be essential for the autonomous replication of R1000. From this analysis, we could predict the origin of replication, promotors and structural genes which regulate the replication of the plasmid. In the coming year, we plan to do extensive genetics on R100, by isolating mutants defective in replication and characterizing them by complementation analysis to confirm the structural and regulatory genes encoded by R100. In the past year, we have also identified and characterized the RNA transcripts and proteins synthesized from the essential region for the replication of R100. Therefore analysis of the nucleotide sequences of mutants having particular cis-dominant mutations and analysis of the transcripts and proteins will further clarify the arrangement of genes on the determined nucleotide sequences of the parent. In the coming year, we will establish an in vitro system of replication of the plasmid. The in vitro system will have great advantages to follow the specific steps of the biochemical reactions involved in replication. We wish to use this system to analyze the roles of the plasmid proteins in replication of this plasmid. Using mutant plasmids to be isolated, we expect to perform complementation analysis in vitro for plasmid DNA replication. Our proposed systematic research research on the replication of the R100 plasmid will elucidate many of the molecular mechanisms involved in the replication of this plasmid.